Application of SSR (Simple Sequence Repeat) marker in identification of authenticity / purity of cucumber variety

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an SSR marker in authenticity / purity identification of a cucumber variety Jingyuanqiv No.2. The SSR primer group for identifying the authenticity / purity of the cucumber variety Jingyuanqiv No.2 comprises at least one pair of eight primer pairs. The invention discloses a method for identifying the authenticity / purity of a cucumber variety Jingyuanqiv No.2. The method comprises the following steps: firstly, extracting genome DNA of a sample to be detected; taking the genome DNA as a template, carrying out PCR (Polymerase Chain Reaction) amplification reaction by utilizing the primer pair to obtain a PCR amplification product, and detecting and judging the variety of the sample to be detected. The SSR primer combination disclosed by the invention can be used for early identification of the cucumber variety Jingyuanqiv No.2 in a seed or seedling stage, and the identification method is rapid, accurate and stable.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of SSR markers in authenticity / purity identification of the cucumber variety Jingyan Chunqiulu No. 2. Background Art

[0002] Cucumber (Cucumis sativus L.) is an annual vine or climbing herb of the Cucurbitaceae family. Cucumbers can be eaten raw or fried, and pickled and processed for a unique flavor. Cucumbers are rich in nutrients such as protein, phosphorus, calcium, iron, vitamin C, carotene, etc., which are not only nutritious but also have health benefits. Fresh cucumbers contain cucumber enzymes that can promote the body's metabolism and have the effect of removing spots and rejuvenating the skin; cucumber vines can also be used as medicine to help regulate blood pressure levels and lower cholesterol. After long-term genetic improvement and directional breeding, cucumber varieties and cultivation types have been differentiated into rich and diverse varieties to adapt to the ecological environment, climate types and consumption habits of different cultivation areas. With the renewal of modern agricultural biotechnology, breeding methods and means are also constantly improving and optimizing. The number of superior cucumber varieties has increased dramatically, and the marketization of the seed industry has increased day by day. Cucumbers have gradually developed into one of the most widely cultivated vegetables in the world, with important agricultural, biological and economic value.

[0003] Variety purity is an important indicator for evaluating seed quality and seed grading, and is directly related to the yield, quality and economic value of agricultural products. The standardization of seed production by seed farmers and the isolation measures of seed production bases will directly affect the purity of seeds. At present, the phenomenon of homonymous or homonymous is common in production, and the illegal production and sale of counterfeit and imitation varieties occurs from time to time. The problem of counterfeiting and infringement is still rampant, causing great economic losses to breeders, seed dealers and farmers. Many dealers sample and test seeds before selling to ensure the authenticity and variety purity of seeds. The field plot planting identification method based on the agronomic traits of plants or fruits is simple, intuitive and easy to observe, but it is greatly affected by environmental conditions and cultivation measures, has a long identification cycle, and requires high technical level of identification personnel. The rapidly developing DNA molecular marker technology provides a possibility for rapid identification of variety purity. SSR technology stands out among many molecular markers with its advantages such as uniform distribution in the genome, co-dominant inheritance, abundant quantity, high polymorphism and strong stability. It is widely used in the construction of core germplasm of cotton, corn, cabbage, Chinese cabbage, genetic diversity analysis, fingerprint map construction, gene positioning of various plants, and hybrid purity identification.

[0004] The new hybrid cucumber variety, Jingyan Chunqiulu No. 2, is a main vine fruit type. It is early-maturing, has a fast fruit expansion rate, a full growth period of about 120 days, is not prone to premature aging, has strong plant growth, has a shiny green skin, moderate nodules, a light green flesh, a fruit length of about 33 cm, a short handle, straight fruit strips, crisp flesh, and a strong flavor. It has strong comprehensive disease resistance, and has both low temperature and weak light tolerance and heat resistance. It is suitable for spring and autumn protected areas and spring and autumn open field cultivation in North China, Northeast China, Northwest China, East China and other regions. The best sowing and planting period can be scientifically and reasonably selected in combination with local climate characteristics, planting methods and supply periods. In order to improve the plant's stress resistance, it is recommended to use high-quality cucumber rootstocks for grafting and seedling raising, which can extend the harvest period and increase the brightness of the fruit. Apply sufficient basal fertilizer before planting, and focus on applying high-quality phosphorus and potassium fertilizers in the middle and late stages. In order to ensure the sowing quality of commercial seeds, a new method is needed to identify the authenticity and purity of cucumber varieties. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides an application of SSR markers in authenticity / purity identification of cucumber variety Jingyan Chunqiulu No. 2.

[0006] An SSR primer set for identifying the authenticity / purity of cucumber variety Jingyan Chunqiulu No. 2, comprising at least one of the following 8 pairs of primers:

[0007] SM22 Primer Pair:

[0008] Forward primer: 5'-TTGAATTCCTCTGCCCAATC-3' (sequence 1 in the sequence listing);

[0009] Reverse primer: 5'-TGGAATTTTGTTAGGGGGAA-3' (sequence 2 in the sequence listing);

[0010] SM24 Primer Pair:

[0011] Forward primer: 5'-CATTCTAGGTCAATGAATCGCA-3' (sequence 3 in the sequence listing);

[0012] Reverse primer: 5'-GCAAAGTTGCCACATTGAAG-3' (sequence 4 in the sequence listing);

[0013] SM33 Primer Pair:

[0014] Forward primer: 5'-TTGGAAAAGTCGCCAAACTT-3' (sequence 5 in the sequence listing);

[0015] Reverse primer: 5'-TCCATGTCTGCTTTTGATTCC-3' (sequence 6 in the sequence listing);

[0016] SM32 Primer Pair:

[0017] Forward primer: 5'-GGCAAATTCCACAGAGCTTC-3' (sequence 7 in the sequence listing);

[0018] Reverse primer: 5'-TACGCCCGATATTCTTCGTC-3' (sequence 8 in the sequence listing);

[0019] SM48 Primer Pair:

[0020] Forward primer: 5'-CCGAGGATACCCATCTCTGA-3' (sequence 9 in the sequence listing);

[0021] Reverse primer: 5'-AGAAGAACACCTGGAACAGACA-3' (sequence 10 in the sequence listing);

[0022] SM53 Primer Pair:

[0023] Forward primer: 5'-ACCAACTTGTGAAAGGGCAT-3' (sequence 11 in the sequence listing);

[0024] Reverse primer: 5'-CACATGATTCACTTCCTTTCATTT-3' (sequence 12 in the sequence listing);

[0025] SM50 Primer Pair:

[0026] Forward primer: 5'-CCCTGCAGCTCTTCTTTGTC-3' (sequence 13 in the sequence listing);

[0027] Reverse primer: 5'-TGGTTACAGAAGCAAAAGGGA-3' (sequence 14 in the sequence listing);

[0028] SM54 Primer Pair:

[0029] Forward primer: 5'-AAACGTCGATCGAGAGAGGA -3' (sequence 15 in the sequence listing);

[0030] Reverse primer: 5'-AAAGAACCGCCATGTCTTTG-3' (sequence 16 in the sequence listing).

[0031] A kit for identifying the authenticity / purity of cucumber variety Jingyan Chunqiulu No. 2, comprising at least one pair of the primer pairs mentioned above.

[0032] A method for authenticating the authenticity / purity of a cucumber variety, Jingyan Chunqiulu No. 2, comprises the following steps:

[0033] Extract the genomic DNA of the sample to be tested; use the genomic DNA as a template, use at least one pair of the primer pairs mentioned above, or the kit mentioned above to perform a PCR amplification reaction to obtain a PCR amplification product; detect the PCR amplification product to determine the variety of the sample to be tested.

[0034] The primer set, and / or the kit, and / or the identification method described above are used to identify the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention provides an SSR molecular marker identification method that can quickly, accurately and stably detect the authenticity / purity of varieties. The SSR primer combination of the present invention can be used for early identification of the cucumber variety Jingyan Chunqiulu No. 2 at the seed or seedling stage, and can also be used for authenticity identification throughout the life cycle starting from the seed to ensure the authenticity of the variety, solve the problem of homonymous and heteronymous varieties in the domestic seed and seedling market, and effectively protect the rights and interests of producers and breeders.

[0037] 2. The electrophoresis bands amplified by PCR in the identification method of the present invention are of moderate size, and the molecular weight difference between two target fragments amplified by the same primer pair is moderate, which is easy to be detected and identified by the operator.

[0038] 3. The identification method of the present invention is based on DNA detection, is not affected by the external environment, and will not change due to changes in environmental conditions. The results are stable and reliable. The identification method of the present invention is used to identify cucumber varieties in various phenological stages, and the results are stable and consistent.

[0039] 4. The method provided by the present invention has the advantages of accuracy, low cost, simple operation, saving manpower and material resources, etc., and has a very broad application prospect.

[0040] 5. The inventors' research group of the present invention applied SSR molecular marker technology, supplemented by field planting comparison verification, and has cumulatively identified more than 3,000 kg of commercial varieties for purity, saving resources, saving manpower and material resources, and greatly shortening the time from harvesting to sales, thereby improving efficiency. At the same time, it provides more accurate technical guidance for the authenticity identification, purity detection, and parental purification of the Jingyan Spring and Autumn Green No. 2 hybrid, and can also provide information for genetic diversity analysis, important phenotypic gene location analysis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is the fingerprint of the polymorphic primers SM32, SM48, SM53 and SM33 in Example 1 on Jingyan Chunqiulu No. 2 (i.e., the electrophoresis diagram of the PCR amplification reaction products). Columns 1-6 are the amplification products of primer pair SM32, columns 1-2 are the male parent, columns 3-4 are the female parent, and columns 5-6 are the F1 hybrid; columns 7-12 are the amplification products of primer pair SM48, columns 7-8 are the male parent, columns 9-10 are the female parent, and columns 11-12 are the F1 hybrid; columns 13-18 are the amplification products of primer pair SM53, columns 13-14 are the male parent, columns 15-16 are the female parent, and columns 17-18 are the F1 hybrid; columns 19-24 are the amplification products of primer pair SM33, columns 19-20 are the male parent, columns 21-22 are the female parent, and columns 23-24 are the F1 hybrid; column M is 500bp DNA Ladder, the bands from top to bottom are 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, and 50bp respectively.

[0042] Figure 2 This is the fingerprint of the polymorphic primers SM22, SM24, SM50 and SM54 in Example 1 on Jingyan Chunqiulu No. 2 (i.e., the electrophoresis diagram of the PCR amplification reaction products). Columns 1-6 are the amplification products of primer pair SM22, columns 1-2 are the male parent, columns 3-4 are the female parent, and columns 5-6 are the F1 hybrid; columns 7-12 are the amplification products of primer pair SM24, columns 7-8 are the male parent, columns 9-10 are the female parent, and columns 11-12 are the F1 hybrid; columns 13-18 are the amplification products of primer pair SM50, columns 13-14 are the male parent, columns 15-16 are the female parent, and columns 17-18 are the F1 hybrid; columns 19-24 are the amplification products of primer pair SM54, columns 19-20 are the male parent, columns 21-22 are the female parent, and columns 23-24 are the F1 hybrid; column M is 500bp DNA Ladder, the bands from top to bottom are 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, and 50bp respectively.

[0043] Figure 3 This is the amplification pattern (i.e., the electrophoresis pattern of the PCR amplification reaction product) of the primer pair SM33 on Jingyan Chunqiulu No. 2 and its parents in Example 2. The first column is the male parent, the second column is the female parent, and the 3rd to 24th columns are F1 hybrids; the Mth column is a 500bp DNA Ladder, and the bands from top to bottom are 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, and 50bp, respectively.

[0044] Figure 4This is the amplification pattern (i.e., the electrophoresis pattern of the PCR amplification reaction product) of the primer pair SM48 on Jingyan Chunqiulu No. 2 and its parents in Example 2. The first column is the male parent, the second column is the female parent, and the 3rd to 24th columns are F1 hybrids; the Mth column is a 500bp DNA Ladder, and the bands from top to bottom are 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, and 50bp, respectively.

[0045] Figure 5 The amplification patterns (i.e., electrophoresis patterns of PCR amplification reaction products) of the primer pairs SM20, SM21, SM35, SM23, SM39, and SM25 on Jingyan Chunqiu Green No. 2 and its parents in Comparative Example 1 are shown in Table 1. Columns 1-4 are the amplification results of SM20, with column 1 being the male parent, column 2 being the female parent, and columns 3-4 being the F1 generation hybrids; columns 5-8 are the amplification results of SM21, with column 5 being the male parent, column 6 being the female parent, and columns 7-8 being the F1 generation hybrids; columns 9-12 are the amplification results of SM35, with column 9 being the male parent, column 10 being the female parent, and columns 11-12 being the F1 generation hybrids; columns 13-16 are the amplification results of SM21, with column 1 being the male parent, column 10 being the female parent, and columns 11-12 being the F1 generation hybrids. 3, column 13 is the male parent, column 14 is the female parent, and columns 15-16 are F1 hybrids; columns 17-20 are the amplification results of SM39, column 17 is the male parent, column 18 is the female parent, and columns 19-20 are F1 hybrids; columns 21-24 are the amplification results of SM25, column 21 is the male parent, column 22 is the female parent, and columns 23-24 are F1 hybrids; column M is a 500bp DNA Ladder, and the bands from top to bottom are 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, and 50bp, respectively. DETAILED DESCRIPTION

[0046] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0047] Cucumber variety authenticity: essentially refers to the true correspondence between a batch of cucumber seeds or plants and their genetic background; in actual work, whether a sample for inspection is authentic refers to whether the genetic characteristics of the sample for inspection are consistent with the document records (such as variety instructions, labels, etc.).

[0048] Cucumber variety purity: The percentage of samples of a specific variety that are identified as authentic (i.e., confirmed to be of this variety) to the total number of samples to be tested. For example, when identifying seeds, in order to distinguish between seeds of non-parental varieties (seeds of non-parental varieties that are authentically identified), male seeds, female seeds, and seeds of this variety, it is necessary to observe, count, and calculate the percentage of variety purity by each seed of the sample to be tested, and use the following formula to calculate:

[0049] Variety purity (%) = {number of seeds for testing - number of seeds of non-parental varieties - number of seeds of male parent - number of seeds of female parent} / number of seeds for testing × 100%;

[0050] Or: Variety purity (%) = number of seeds of this variety / number of seeds for testing × 100%.

[0051] The new cucumber variety "Jingyan Chunqiulu No. 2" of the present invention is a cucumber variety cultivated by the research team of the inventor of the present invention. Anyone can freely obtain the variety "Jingyan Chunqiulu No. 2" for the purpose of achieving the purpose of the present invention. The contact information is: Address: No. 50, Zhanghua Road, Haidian District, Beijing, Tel: 010-51503023. This variety can also be purchased on the market.

[0052] In a first aspect of the present invention, there is provided an SSR primer set for identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2, comprising at least one of the following 8 pairs of primers:

[0053] SM22 Primer Pair:

[0054] Forward primer: 5′-TTGAATTCCTCTGCCCAATC-3′ (sequence 1);

[0055] Reverse primer: 5′-TGGAATTTTGTTAGGGGGAA-3′ (sequence 2);

[0056] SM24 Primer Pair:

[0057] Forward primer: 5′-CATTCTAGGTCAATGAATCGCA-3′ (SEQ ID NO: 3);

[0058] Reverse primer: 5′-GCAAAGTTGCCACATTGAAG-3′ (SEQ ID NO: 4);

[0059] SM33 Primer Pair:

[0060] Forward primer: 5′-TTGGAAAAGTCGCCAAACTT-3′ (SEQ ID NO: 5);

[0061] Reverse primer: 5′-TCCATGTCTGCTTTTGATTCC-3′ (SEQ ID NO: 6);

[0062] SM32 Primer Pair:

[0063] Forward primer: 5′-GGCAAATTCCACAGAGCTTC-3′ (SEQ ID NO: 7);

[0064] Reverse primer: 5′-TACGCCCGATATTCTTCGTC-3′ (SEQ ID 8);

[0065] SM48 Primer Pair:

[0066] Forward primer: 5′-CCGAGGATACCCATCTCTGA-3′ (SEQ ID NO: 9);

[0067] Reverse primer: 5′-AGAAGAACACCTGGAACAGACA-3′ (SEQ ID NO: 10);

[0068] SM53 Primer Pair:

[0069] Forward primer: 5′-ACCAACTTGTGAAAGGGCAT-3′ (SEQ ID NO: 11);

[0070] Reverse primer: 5′-CACATGATTCACTTCCTTTCATTT-3′ (SEQ ID NO: 12);

[0071] SM50 Primer Pair:

[0072] Forward primer: 5′-CCCTGCAGCTCTTCTTTGTC-3′ (SEQ ID NO: 13);

[0073] Reverse primer: 5′-TGGTTACAGAAGCAAAAGGGA-3′ (SEQ ID NO: 14);

[0074] SM54 Primer Pair:

[0075] Forward primer: 5′-AAACGTCGATCGAGAGAGGA-3′ (SEQ ID NO: 15);

[0076] Reverse primer: 5'-AAAGAACCGCCATGTCTTTG-3' (SEQ ID NO: 16).

[0077] In a second aspect of the present invention, a kit for identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2 is provided, the kit comprising at least one pair of the eight pairs of primers described in the second aspect of the present invention.

[0078] Preferably, the kit further comprises: at least one of a PCR amplification buffer, dNTPs, Taq enzyme and a DNA color developing dye.

[0079] In a third aspect, the present invention provides a method for identifying the authenticity / purity of a cucumber variety, Jingyan Chunqiulu No. 2. The method comprises using the genomic DNA of a sample to be tested as a template, and using the SSR primer set for identifying the authenticity / purity of a cucumber variety, Jingyan Chunqiulu No. 2, provided in the first aspect of the present invention, or the kit for identifying the authenticity / purity of a cucumber variety, Jingyan Chunqiulu No. 2, provided in the second aspect of the present invention, to perform a PCR operation.

[0080] In a preferred embodiment, the method comprises the following steps:

[0081] S1: Extract genomic DNA of the sample to be tested.

[0082] When the sample to be tested is a seed, it is preferred to use young cotyledons germinated from the seeds as test materials, and the SDS method is preferably used to extract genomic DNA; when the sample to be tested is a plant, it is preferred to use leaves as test materials, and the CTAB method is preferably used to extract genomic DNA.

[0083] S2: Using the genomic DNA of the sample to be tested as a template, performing a PCR amplification reaction using at least one pair of primers described in the first aspect of the present invention, or the kit described in the second aspect of the present invention, to obtain a PCR amplification product.

[0084] The following PCR amplification reaction system (20 μL system) is preferably used:

[0085] Template DNA (concentration 50 ng / μL) 1 μL, 10× Taq buffer 2 μL, 10× color dye 2 μL, 10 mM dNTP 0.4 μL, 2.5 U Taq DNA polymerase 0.3 μL, 0.5 μM forward primer 1 μL, 0.5 μM reverse primer 1 μL, add ultrapure water to make up to 20 μL.

[0086] The following PCR amplification reaction program is preferably used:

[0087] First, pre-denaturation was performed at 94°C for 4 min; then, denaturation was performed at 94°C for 15 s, annealing was performed at 55°C for 20 s, and extension was performed at 72°C for 30 s, for a total of 35 cycles; then, extension was performed at 72°C for 5 min and the samples were stored at 4°C.

[0088] S3: Detect the PCR amplification products of each sample to be tested to determine whether the sample to be tested is Jingyan Chunqiulu No. 2.

[0089] The above detection method preferably uses electrophoresis to detect the PCR amplification product; the electrophoresis can be agarose gel electrophoresis or polyacrylamide gel electrophoresis.

[0090] Specifically:

[0091] If it is detected that the above-mentioned SM22 primer pair only amplifies a target band of 193bp in size, the sample to be tested is the father; if only a target band of 175bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental hybrid.

[0092] If it is detected that the above-mentioned SM24 primer pair only amplifies a target band of 206bp in size, the sample to be tested is the father; if only a target band of 183bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental variety.

[0093] If it is detected that the above-mentioned SM33 primer pair only amplifies a target band of 152bp in size, the sample to be tested is the father; if only a target band of 177bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental hybrid.

[0094] If it is detected that the above-mentioned SM32 primer pair only amplifies a target band of 231bp in size, the sample to be tested is the father; if only a target band of 246bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental variety.

[0095] If it is detected that the above-mentioned SM48 primer pair only amplifies a target band of 212bp in size, the sample to be tested is the father; if only a target band of 185bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental variety.

[0096] If it is detected that the above-mentioned SM53 primer pair only amplifies a target band of 216bp in size, the sample to be tested is the father; if only a target band of 198bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental variety.

[0097] If it is detected that the above SM50 primer pair only amplifies a target band of 155bp in size, the sample to be tested is the father; if only a target band of 172bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental hybrid.

[0098] If it is detected that the above-mentioned SM54 primer pair only amplifies a target band of 153bp in size, the sample to be tested is the father; if only a target band of 137bp in size is amplified, the sample to be tested is the mother; if the above two target bands are amplified at the same time, the sample to be tested is an F1 hybrid (i.e., variety Jingyan Chunqiulu No. 2); if none of the above target bands are amplified, the sample to be tested is a non-parental hybrid.

[0099] The fourth aspect of the present invention provides the use of the SSR primer set described in the first aspect, and / or the SSR kit described in the second aspect, and / or the method described in the third aspect in identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2.

[0100] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.

[0101] Example 1

[0102] This example is used to describe a method for obtaining an SSR primer set for identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2.

[0103] Experimental materials: Parents of the new variety Jingyan Chunqiulu No. 2 and its F1 hybrid bred by the cucumber development team of the Vegetable Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0104] 1. Genomic DNA extraction and quality testing

[0105] The experiment was conducted in the Variety Purity Molecular Identification Laboratory of the Vegetable Research Institute of Beijing Academy of Agricultural and Forestry Sciences in December 2023. In accordance with the provisions of GB / T 3543.5, the seeds were placed in a germination box covered with filter paper for germination. When the seedlings grew to the cotyledons, the young cotyledons were picked, and the genomic DNA was extracted by SDS extraction. The integrity, concentration and quality of the DNA were tested by a spectrophotometer and stored at -20°C for later use.

[0106] For the above-mentioned SDS extraction method for extracting genomic DNA, please refer to: Wang Shen, Fan Baojie, Liu Changyou, Wang Yan, Shi Huiying, Zhang Zhixiao, Su Qiuzhu, Shen Yingchao, Tian Jing, Analysis of the optimal extraction method of genomic DNA from mung bean callus, [J / OL] Molecular Plant Breeding, https: / / link.cnki.net / urlid / 46.1068.s.20240726.0922.002.

[0107] 2. SSR Primer Screening

[0108] 1. 74 sets of SSR primer pairs were synthesized and screened by Shanghai Sixing Biotechnology Co., Ltd.

[0109] 2. Mix 10 DNA samples of Jingyan Chunqiulu No. 2 and its parents into 2 groups of samples and perform PCR amplification reaction.

[0110] The 20 μL PCR reaction system included: 1 μL template DNA (concentration 50 ng / μL), 2 μL 10× Taq buffer, 2 μL 10× dye, 0.4 μL 10 mM dNTP Mixture, 0.3 μL 2.5 U Taq DNA Polymerase (Beijing Huitian Oriental Technology Co., Ltd.), 1 μL 0.5 μM Forward Primer, 1 μL 0.5 μM Reverse Primer, and make up with ultrapure water.

[0111] The PCR reaction cycle was set as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0112] 3. Agarose gel electrophoresis detection:

[0113] Electrophoresis was performed at 200W for 60 min, and PCR products were separated by 2.3% agarose gel. The raw data were collected by photographing under UV light in a G:BOX Chemi-XRQ image analysis system (Gene Biotechnology International Trade (Shanghai) Co., Ltd.). Primers with complementary parental banding patterns and hybrids with both parental banding patterns were selected to perform single detection on young cotyledon genomic DNA.

[0114] 4. Results and analysis:

[0115] By observing and analyzing the band patterns of the electrophoresis bands of the amplified products, it was found that the SSR amplified fragments of the Beijing Research Spring and Autumn Green No. 2 hybrid were distributed between 130-270bp, most of the primers amplified 1-2 sites, some were 3, and most of the sites were concentrated in the range of 160-220bp. High-resolution agarose gel electrophoresis imaging showed that among the 74 sites, the amplified products of F1 at 9 sites contained parental band patterns and were complementary, which can be used for the purity detection of hybrid seeds. These 9 pairs of SSR primers constructed the fingerprint map of Beijing Research Spring and Autumn Green No. 2 (Table 1). Among them, 8 pairs of primers: SM22, SM24, SM33, SM32, SM48, SM53, SM50, SM54 ( Figure 1 and Figure 2 ) The amplified products have clear bands and similar brightness, and the molecular weight difference of the fragments is greater than 10bp, which is easy to distinguish. Although primer SM26 is also polymorphic and can be stably amplified, the band clarity is low and it is impossible to accurately distinguish hybrids, heteromorphic strains (i.e., non-parental varieties) and parents. Therefore, the present invention selects SM22, SM24, SM33, SM32, SM48, SM53, SM50, and SM54, these 8 pairs of SSR primers for authenticity / purity identification of the cucumber variety Jingyan Chunqiulu No. 2.

[0116] Table 1: Polymorphic SSR primers and their sequences

[0117]

[0118] Example 2

[0119] This example is used to describe a method for identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2 using primer pairs SM33 and SM48. The method comprises the following steps:

[0120] S1. Field plot planting

[0121] In the 7 greenhouses (numbered A, B, C, D, E, F, and G) of 7 farmers in the seed production base, 100 Jingyan Spring and Autumn Green No. 2 F1 hybrid seeds were randomly sown in each greenhouse, with a row spacing of 40-50 cm and protection rows set up around them, and field management was carried out according to routine procedures.

[0122] S2: Extraction of genomic DNA from samples to be tested

[0123] According to the method described in Example 1, the seeds were germinated until the cotyledons unfolded, and the young cotyledons were picked. Genomic DNA was extracted using the SDS extraction method according to the method described in Example 1 and stored at -20°C for later use.

[0124] S3: PCR amplification reaction

[0125] The genomic DNA of the sample to be tested, the genomic DNA of the parents and the genomic DNA of another cucumber variety Jingyan Xiamei were used as templates, and primer pairs SM33 and SM48 were used respectively. PCR amplification reactions were performed according to the reaction system and reaction procedure of Example 1 to obtain PCR amplification products of SM33 and SM48.

[0126] S4: Agarose gel electrophoresis detection:

[0127] The PCR amplification product was detected by referring to the method of Example 1, and the results are as follows.

[0128] like Figure 3 The electrophoresis spectrum of the amplified products showed that in the amplified products of primer pair SM33, the paternal parent in the first column could amplify a band of 152bp, the maternal parent in the second column could amplify a band of 177bp, and the F1 hybrids in columns 3-24 could simultaneously amplify two bands of 152 and 177bp, indicating that the authenticity of the samples in columns 3-24 was identified as the Jingyan Chunqiulu No. 2 variety.

[0129] like Figure 4 The electrophoresis spectrum of the amplified products showed that in the amplified products of primer pair SM48, the paternal parent in the first column could amplify a band of 212bp, the maternal parent in the second column could amplify a band of 185bp, and the F1 hybrids in columns 3-24 could simultaneously amplify two bands of 212 and 185bp, indicating that the authenticity of the samples in columns 3-24 was identified as the Jingyan Chunqiulu No. 2 variety.

[0130] None of the above target bands were detected in the amplified products of the above-mentioned Jingyan Xiamei cucumber variety.

[0131] S5: Variety purity calculation results:

[0132] According to the formula: Variety purity (%) = {number of seeds for testing - number of seeds of non-parental heterogeneous varieties - number of seeds of the male parent - number of seeds of the female parent} / number of seeds for testing × 100%, that is, the percentage of the number of seeds of this variety to the number of seeds of this crop sample for testing, the seedling purity (i.e., variety purity) in greenhouse C was 97%, the seedling purity in greenhouse E was 99%, and the seedling purity in greenhouses A, B, D, F, and G was 100%. The results of the agronomic trait identification of field planting in the future showed that the agronomic traits of 2 of the 4 pseudohybrids were significantly different from those of Jingyan Chunqiulu No. 2 and its parents, and were judged to be other non-parental heterogeneous varieties; the other 2 were consistent with the characteristics of the female parent and were judged to be self-pollinated varieties of the female parent; this result was consistent with the results of SSR molecular identification.

[0133] Example 3

[0134] This example is used to describe a method for identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2 using primer pairs SM22, SM24, SM32, SM53, SM50, and SM54.

[0135] In this embodiment, the genomic DNA of the sample to be tested in Example 2, the genomic DNA of the parents and the genomic DNA of another cucumber variety Jingyan Xiamei were used as templates. According to the method of Example 1, PCR amplification reactions were performed using primer pairs SM22, SM24, SM32, SM53, SM50, and SM54 to obtain amplification products respectively.

[0136] Each amplification product was tested according to the method of Example 1, and the results are as follows:

[0137] In the amplification products of primer pair SM22, it can be seen that the male parent can amplify a 193 bp band, the female parent can amplify a 175 bp band, and the F1 hybrid can amplify the above two bands at the same time.

[0138] In the amplification products of primer pair SM24, it can be seen that the male parent can amplify a band of 206 bp, the female parent can amplify a band of 183 bp, and the F1 hybrid can amplify the above two bands at the same time.

[0139] In the amplification products of primer pair SM32, it can be seen that the male parent can amplify a band of 231 bp, the female parent can amplify a band of 246 bp, and the F1 hybrid can amplify the above two bands at the same time.

[0140] In the amplification products of primer pair SM53, it can be seen that the male parent can amplify a band of 216 bp, the female parent can amplify a band of 198 bp, and the F1 hybrid can amplify the above two bands at the same time.

[0141] In the amplification products of primer pair SM50, it can be seen that the male parent can amplify a band of 155 bp, the female parent can amplify a band of 172 bp, and the F1 hybrid can amplify the above two bands at the same time.

[0142] In the amplification products of primer pair SM54, it can be seen that the male parent can amplify a 153 bp band, the female parent can amplify a 137 bp band, and the F1 hybrid can amplify the above two bands at the same time.

[0143] None of the above target bands were detected in the amplified products of the above-mentioned Jingyan Xiamei cucumber variety.

[0144] Then calculate according to the following formula: Variety purity (%) = {number of seeds for testing - number of seeds of non-parental varieties - number of seeds of male parent - number of seeds of female parent} / number of seeds for testing × 100%, and conclude that the seedling purity (i.e., variety purity) in greenhouse C is 97%, the seedling purity in greenhouse E is 99%, and the seedling purity in greenhouses A, B, D, F, and G is 100%. This result is consistent with the identification of agronomic traits in field planting in the future (a total of 4 pseudohybrids, 2 of which are non-parental varieties and 2 are self-pollinated varieties of the female parent), indicating that the SSR identification method is accurate and reliable.

[0145] Comparative Example 1

[0146] This comparative example is used to describe the method for identifying the authenticity of cucumber variety Jingyan Chunqiulu No. 2 using primer pairs SM20, SM21, SM35, SM23, SM39 and SM25 that failed to pass the screening among the 74 candidate primer pairs in Example 1. The sequence composition of the above primer pairs is as follows:

[0147] SM20 Primer Pair:

[0148] Forward primer: 5′-CCGTTGGTCTTGGACTCTCA-3′;

[0149] Reverse primer: 5′-TGTAAAAGTGATCAGGAGGGTCT-3′;

[0150] SM21 Primer Pair:

[0151] Forward primer: 5′-CACAATCCCACGAAGAACAA-3′;

[0152] Reverse primer: 5′-TGCAATTATGGCAAATCAAAA-3′;

[0153] SM35 Primer Pair:

[0154] Forward primer: 5′-GAGCCATCCATCAGAGAGAGA-3′;

[0155] Reverse primer: 5′-ACCCACAAGCTTCAGAGGTC-3′;

[0156] SM23 Primer Pair:

[0157] Forward primer: 5′-TGGCTTTTCTGTCACGTCC-3′;

[0158] Reverse primer: 5′-TCCATGGTACAACAAGAATCACA-3′;

[0159] SM39 Primer Pair:

[0160] Forward primer: 5′-ATCCAAGAGGTTTCCAACGA-3′;

[0161] Reverse primer: 5′-TGGTCAATGAGCTTAGCTTTCTC-3′;

[0162] SM25 Primer Pair:

[0163] Forward primer: 5′-GCTAACACTACCCGCTGCTT-3′;

[0164] Reverse primer: 5′-AACAGAAAGAGAATCGGGGG-3′.

[0165] The templates for the PCR amplification reaction in this comparative example are the genomic DNA of cucumber, which has been identified as Jingyan Chunqiulu No. 2 by primers SM33 and SM48, and the genomic DNA of the parents. This comparative example uses primer pairs SM20, SM21, SM35, SM23, SM39 and SM25 for PCR amplification reaction. The test results are as follows Figure 5 As shown. It can be seen that in the amplified bands of these six primer pairs, there is no difference in the fragment size of the target bands of the male and female parents, and the two target bands of the F1 hybrid cannot detect the difference in fragment size and cannot be clearly distinguished. Therefore, these six primer pairs are not suitable as SSR primers for identifying the authenticity of the cucumber variety or seed purity of Jingyan Chunqiulu No. 2.

[0166] Comparative Example 2

[0167] This comparative example is used to describe the method of using the 8 sets of primer pairs of the present invention to identify the authenticity of the cucumber variety Jingyan Chunqiulu No. 2.

[0168] The templates for the PCR amplification reaction in this comparative example are the genomic DNA of three non-parental varieties that are relatively close to the genetic background of Jingyan Chunqiulu No. 2 - cucumber varieties Jingyan Xiamei, Jingyan Xiamei No. 2, and Jingyan Chunqiulu. This comparative example uses the eight pairs of primers of the present invention to carry out PCR amplification reactions, and the test results show that none of the aforementioned target bands amplified by any of the eight pairs of primers were detected in the PCR amplification products of the three varieties of cucumbers. It can be seen that the eight pairs of primers of the present invention cannot detect any of the aforementioned target bands when detecting cucumber varieties other than Jingyan Chunqiulu No. 2 and its parents; therefore, the identification method of the present invention can clearly distinguish Jingyan Chunqiulu No. 2 from other cucumber varieties with similar genetic backgrounds.

[0169] Variety purity identification is an important indicator for accurately assessing seed grades, and has high application value in variety registration, variety rights protection, parent purification and genetic diversity research. At present, my country is at the forefront of the world in cucumber breeding, whether it is conventional means or molecular technology and gene editing, but only various departments or research units are leading in different breeding stages, lacking close contact and efficient collaboration among all departments in the whole process. Seed production is seriously decentralized, small-scale and not highly standardized, and the quality and yield are unstable. The biggest problem in the sales link is that the protection of intellectual property rights is not strong enough, and infringements such as counterfeiting and imitation occur from time to time, causing huge economic losses to seed dealers and farmers, and seriously damaging the innovation enthusiasm of property rights units. In order to ensure the genetic stability of commercial seeds, seeds need to be sampled and tested for purity before they are sold.

[0170] Since field purity identification consumes a lot of manpower and material resources, and the identification cycle is long, it has been gradually replaced by indoor DNA molecular marker methods. Especially for commercial seeds that are in a hurry to be put on the market, DNA molecular marker identification of variety purity has become an indispensable part of cucumber seed production and sales. With the continuous improvement of DNA molecular marker identification technology in experimental procedures and detection systems, it has become increasingly procedural, standardized, and efficient. The International Union for the Protection of New Varieties of Plants (UPOV) has included it in the scope of DUS testing of crop varieties.

[0171] Among the 74 pairs of SSR primers selected by the present invention, 9 pairs are polymorphic and co-dominant in the PCR amplification products of the parent and hybrid F1 of cucumber Jingyan Spring and Autumn Green No. 2, forming the SSR fingerprint of the Jingyan Spring and Autumn Green No. 2 F1 hybrid and the parent. Among them, the polymorphic fragments amplified by 8 pairs of primers SM22, SM24, SM33, SM32, SM48, SM53, SM50, and SM54 have obvious molecular weight differences, good stability, and high band clarity, which are easy to distinguish and identify. The SSR molecular marker purity identification of Jingyan Spring and Autumn Green No. 2 cucumber seeds was compared with the field plot planting identification, and it was found that the two had a good degree of consistency and no obvious result deviation was found. Accurate and efficient SSR molecular marker technology provides technical support for the purity identification, genetic map drawing, variety approval and protection of Jingyan Spring and Autumn Green No. 2 cucumber seeds. Compared with field plot planting identification, it is simpler and more convenient, can promote the timely listing of excellent new varieties, and is not restricted by environmental conditions such as temperature, climate, and soil, saving manpower and material resources. At different positions in the cucumber genome, fragment length polymorphism is formed due to differences in the sequence and number of repeat units at each SSR site in different varieties. When identifying the purity of cucumber hybrids, the reliability of the identification results increases with the number of SSR sites measured. In seed production practice, it was found that hybridization of non-paternal pollen and maternal self-pollination are the main factors that lead to reduced purity of cucumber hybrids. Therefore, the selection of a pair of effective SSR primers can quickly distinguish the mixture of hybrids and parental seeds. In the early stage of the promotion of Jingyan Chunqiulu No. 2, traditional field purity identification was used, which was inefficient and had a long cycle.

[0172] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An SSR primer set for identifying the authenticity / purity of cucumber variety Jingyan Chunqiulu No. 2, characterized in that: Include at least one of the following 8 primer pairs: SM22 Primer Pair: Forward primer: 5′-TTGAATTCCTCTGCCCAATC-3′; Reverse primer: 5′-TGGAATTTTGTTAGGGGGAA-3′; SM24 Primer Pair: Forward primer: 5′-CATTCTAGGTCAATGAATCGCA-3′; Reverse primer: 5′-GCAAAGTTGCCACATTGAAG-3′; SM33 Primer Pair: Forward primer: 5′-TTGGAAAAGTCGCCAAACTT-3′; Reverse primer: 5′-TCCATGTCTGCTTTTGATTCC-3′; SM32 Primer Pair: Forward primer: 5′-GGCAAATTCCACAGAGCTTC-3′; Reverse primer: 5′-TACGCCCGATATTCTTCGTC-3′; SM48 Primer Pair: Forward primer: 5′-CCGAGGATACCCATCTCTGA-3′; Reverse primer: 5′-AGAAGAACACCTGGAACAGACA-3′; SM53 Primer Pair: Forward primer: 5′-ACCAACTTGTGAAAGGGCAT-3′; Reverse primer: 5′-CACATGATTCACTTCCTTTCATTT-3′; SM50 Primer Pair: Forward primer: 5′-CCCTGCAGCTCTTCTTTGTC-3′; Reverse primer: 5′-TGGTTACAGAAGCAAAAGGGA-3′; SM54 Primer Pair: Forward primer: 5′-AAACGTCGATCGAGAGAGGA-3′; Reverse primer: 5'-AAAGAACCGCCATGTCTTTG-3'.

2. A kit for identifying the authenticity / purity of cucumber variety Jingyan Chunqiulu No. 2, characterized in that: The kit comprises at least one of the 8 pairs of primers as claimed in claim 1.

3. The kit according to claim 2, characterized in that: The kit also includes: at least one of a PCR amplification buffer, dNTP, Taq enzyme and a DNA color developing dye.

4. A method for identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2, characterized in that: The method comprises the following steps: Extracting genomic DNA from the sample to be tested; Using the genomic DNA as a template, performing a PCR amplification reaction using at least one of the primer pairs described in claim 1, or the kit described in claim 2 or 3, to obtain a PCR amplification product; The PCR amplification product is detected to determine the type of the sample to be tested.

5. The identification method according to claim 4, characterized in that: The determination method is as follows: If it is detected that the SM22 primer pair only amplifies a target band of 193 bp, the sample to be tested is the male parent; if only a target band of 175 bp is amplified, the sample to be tested is the female parent; if both of the above target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulu No. 2; If none of the above target bands are amplified, the sample to be tested is a non-parental hybrid; If it is detected that the SM24 primer pair only amplifies a target band of 206 bp, the sample to be tested is the male parent; if only a target band of 183 bp is amplified, the sample to be tested is the female parent; if the above two target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulu No. 2; if none of the above target bands are amplified, the sample to be tested is a non-parental alien variety; If it is detected that the SM33 primer pair only amplifies a target band of 152 bp, the sample to be tested is the male parent; if only a target band of 177 bp is amplified, the sample to be tested is the female parent; if the above two target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulv No. 2; if none of the above target bands are amplified, the sample to be tested is a non-parental alien variety; If it is detected that the SM32 primer pair only amplifies a target band of 231 bp, the sample to be tested is the male parent; if only a target band of 246 bp is amplified, the sample to be tested is the female parent; if the above two target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulv No. 2; if none of the above target bands are amplified, the sample to be tested is a non-parental alien variety; If it is detected that the SM48 primer pair only amplifies a target band of 212 bp, the sample to be tested is the male parent; if only a target band of 185 bp is amplified, the sample to be tested is the female parent; if both of the above target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulu No. 2; If none of the above target bands are amplified, the sample to be tested is a non-parental hybrid; If it is detected that the SM53 primer pair only amplifies a target band of 216 bp, the sample to be tested is the male parent; if only a target band of 198 bp is amplified, the sample to be tested is the female parent; if both of the above target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulu No. 2; If none of the above target bands are amplified, the sample to be tested is a non-parental hybrid; If it is detected that the SM50 primer pair only amplifies a target band of 155 bp, the sample to be tested is the male parent; if only a target band of 172 bp is amplified, the sample to be tested is the female parent; if the above two target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulv No. 2; if none of the above target bands are amplified, the sample to be tested is a non-parental alien variety; If it is detected that the SM54 primer pair only amplifies a target band of 153bp in size, the sample to be tested is the father's parent; if only a target band of 137bp in size is amplified, the sample to be tested is the mother's parent; if the above two target bands are amplified at the same time, the sample to be tested is Zhongjingyan Chunqiulv No. 2; if none of the above target bands are amplified, the sample to be tested is a non-parental heterogeneous variety.

6. The identification method according to claim 4 or 5, characterized in that: The 20 μL system of the PCR amplification reaction includes: 1 μL of 50 ng / μL template DNA, 2 μL of 10× Taq buffer, 2 μL of 10× color developing dye, 0.4 μL of 10 mM dNTP, 0.3 μL of 2.5 U Taq DNA polymerase, 1 μL of 0.5 μM forward primer, 1 μL of 0.5 μM reverse primer, and make up with ultrapure water.

7. The identification method according to claim 4 or 5, characterized in that: The procedure of the PCR amplification reaction includes: First, pre-denaturation was performed at 94°C for 4 min; then, denaturation was performed at 94°C for 15 s, annealing was performed at 55°C for 20 s, and extension was performed at 72°C for 30 s, for a total of 35 cycles; then, extension was performed at 72°C for 5 min, and the samples were stored at 4°C.

8. Use of the primer set according to claim 1, and / or the kit according to claim 2 or 3, and / or the identification method according to any one of claims 4 to 7 in identifying the authenticity / purity of the cucumber variety Jingyan Chunqiulu No. 2.

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